Antibiotic derivatives of mitomycins a, b, c and porfiromycin



United States Patent 3,332,944 ANTIBIOTKC DERHVATEVES 0F MITQMYCINS A,B, C AND PORFIROMYCIN Donna Bernice (Josuiich, Pearl River, James BurnsPatrick, Suifern, and Richard Preston Williams, Tomkins Cove, N.Y.,assignors to American Cyanamid Company, Stamford, (101111., acorporation of Maine No Drawing. Filed Nov. 2, 1964, Ser. No. 408,332

Claims. (Cl. 260--247.2)

This application is a continuation-in-part of our copending applicationSer. No. 250,027 filed Jan. 8, 1963, now abandoned, which in turn is acontinuation-in-part of application Ser. No. 49,176, filed Aug. 12,1960, now abandoned.

This invention relates to a novel series of active antibacterial agentsderived by reaction of certain of the mitomycin group of antibioticswith certain classes of amines. More particularly, this invention isconcerned with novel compounds which may be represented by the followinggeneral formula:

wherein R is selected from the group consisting of phenyl loweralkylamino, N-lower alkyl phenyl lower alkylamino, lower dialkylaminolower alkylarnino, carboxy lower alkylarnino, lower alkyleneimino, cyclolower alkylamino, hydroxy lower alkylamino, pyridylamino, pyridyl lower:alkylamino, quinolylamino, morpholino, piperazino and a moiety of theformula:

@Nla

wherein R is selected from the group consisting of hydrogen, halogen,lower alkyl, lower alkoxy and lower car- 'boalkoxy; R is selected fromthe group consisting of hydrogen and methyl; and R is selected from thegroup consisting of hydrogen, lower alkyl and phenyl lower alkyl.Suitable lower alkyl, lower alkylene, lower alkoxy, lower alkanoyl andlower carboalkoxy groups contemplated by the present invention are thosehaving up to about six carbon atoms. Halogen is exemplified by chlorine,bromine and iodine.

Certain of the starting materials for the preparation of the novelcompounds of this invention are in themselves novel antibiotics producedby cultivation of appropriate strains of Streptomyces verticillatus ATCCNos. 13,495; 13,538 and 13,539. These novel elaboration products,formerly designated B, 'y ,-and 7 are now known, respectively, asmitomycin A, mitomycin B and porfiromycin. Structure elucidation ofmitocycins A, B and C and porfiromycin is described by J. S. Webb et al.in J .A.C.S., 84, 3185 (1962).

Another class of suitable starting materials for the prep :aration ofthe novel products of this invention are the N-substituted derivativesof mitomycin A and mitomycin C which form the subject matter of thecopending application of Meyer et .al., Ser. No. 200,631 filed June 7,1962, now Patent No. 3,226,393.

For convenience, the structures of the mitomycins and porfiromycinreferred to above are reproduced below.

mitomycin C may be represented by the following general formula:

wherein X is lower alkoxy or amino, and R is lower alkyl, loweralkanoyl, phenyl lower alkyl, benzoyl, phenyl carbamoyl, lower alkylsulfonyl, phenyl sulfonyl and lower carboalkoxy.

In accordance with the present invention, we have discovered that it ispossible to prepare highly useful antibacterial agents by reaction ofthe foregoing group of antibiotics with primary and secondary amines.

The novel antibiotics of this invention are, in general, coloredcrystalline solids of relatively limited solubility in water, butsomewhat more soluble in lower alkanols,

. ethyl acetate, acetone, toluene, benzene, chloroform, and

the like.

The amines which may be reacted with the five starting antibiotics areprimary and secondary amines which may be aliphatic (acyclic orisocyclic), aromatic, heterocyclic,

' or mixed (for example: aralkyl, alkaryl, aliphatic-aromatic,aliphatic-heterocyclic, aromatic-heterocyclic, aliphatic-aralkyl, etc.),including polyfunctional amines such as hydroxyalkylamines,haloarylamines, amino acids, and the like. Excluded are tertiary amines,quaternary ammonium compounds, ammonia, and such inorganic substitutionproducts of ammonia as hydroxylarnine, hydrazine, chloroarnine, and thelike.

It is an advantage of this invention that the novel reaction productsmay be prepared for the most part in simple reaction media. Ordinarilywater is used as a reaction medium, but other liquids such as loweralkanols, ethyl acetate, benzene, toluene, chloroform, or the like canbe used.

Typically, the starting material is dissolved in a suitable volume ofwater, and any excess of amine either alone or dissolved in water orother suitable solvent is added with stirring. Very often the progressof the reaction is accompanied by a visible color change or a shift inthe ultra violet absorption spectrum. In general, after the reaction iscompleted or largely completed, the reaction medium and excess reactantmay be removed by vacuum distillation and the product may then berecovered by conventional means such as crystallization, chronatography,and the like. The novel antibiotics of this invention and the startingantibiotics are sensitive to acid and to high temperatures, and inconsequence, care nust be taken to avoid their exposure to these twocon- ;litions. When distillation is employed, for example, to removereaction medium and excess reactant, a high :nough vacuum should bemaintained to keep the temaerature low.

The novel antibiotics of this invention have broad- :pectrum activityagainst a number of microorganisms tnd thus are useful antibacterialagents. The new anti- )iotics show in vitro activity in standard,scientifically 'ecognized microbiological assays, against the following)rganisms:

Klebsiella pneumoniae A strain AD iacz'llus cereus ATCC 10702dycobaczerium smegmatis ATCC 607 treptococcus pyogenes ,8 hemolyticusEscherichia coli ATCC 9637 'almonella gallinarum 'taphylococcus aureusATCC 6538 seudomonas aeruginosa ATCC 10145 treptococcus faecal'z'staphylococcus albus No. 69 treptococcus sp. ,8 hemolyticus No. 80taphylococdus aureus NY 104 'orynebacterium xerose treptococcus sp. ahemolyticus .scherichia coli No.22

acillus subtilis ATCC 6633 llcaligenes faccalis ATCC 10153 roteusvulgaris ATCC 9484 arcina lutea ATCC 9341 The novel antibiotics of thisinvention are active against ram-positive and gram-negativemicroorganisms, such s staphylococci, pneumococci, and streptococci. Theovel compounds are thus potentially useful as therapeuc agents intreating bacterial infections in humans and aimals caused by suchmicroorganisms. The compounds m be usefully employed for controllingsuch infections J topical application or internal administration.

The usefulness of the novel compounds is demonrated by their ability tocontrol systemic lethal infec- Jns in mice. Thus the novel antibioticsof this in- :ntion show very high antibacterial in vivo activity in iceagainst Staphlococcus aureus, strain Smith, rangg by direct comparisonsfrom about /2 to about 8 nes the activity of tetracycline. Although thenovel rtibiotics of this invention have not as yet beenclinicaldemonstrated to be useful in human therapy, the nditions of thetests in mice against human pathogens ow a high probability of usefulactivity in humans.

The invention will be described in greater detail in njunction with thefollowing specific examples.

EXAMPLE 1 1.5 parts of mitomycin A are dissolved in 1,000 parts waterand 100 parts of aniline added with shaking. re purple solution which isthe characteristic color of .tornycin A gradually changes color togreen. When a reaction is complete the reaction mixture is frozen ddried in the frozen state, resulting in a green oil rich contains thecrude antibiotics with the excess unacted aniline. The crude antibioticsare then separated chromatography using a diatomaceous earth packed lumnand a system consisting of 1 part heptane, 4 parts iyl acetate, 3 partsmethanol and 2 parts water. On the lumn, the product appears as a fastmoving green nd. The first fraction from the column is thenrechrottographed and green crystals of the new antibiotic are obtained.When dissolved in various solvents the following absorption bands arenoted:

MES 214, 259, 376 mu; AEQQ 373 my; A313, 262, 373 mp.

The infrared spectrum is as follows: M3; 2.90, 3.40, 5.84, 6.09, 6.38,6.61, 6.90, 7.54, 7.92,

EXAMPLE 2 One part of mitomycin B is dissolved in water and excessethyleneimine is added. The color changes rapidly from purple to paleblue. After one and one-half hours the ultraviolet maximum has moved outto 365 m from the initial position of 320 mg. The solvent and excessreagent are then removed in vacuo to give a new green antibioticresidue. The crude antibiotic is then purified by chromatography orrecrystallized as in Example 1. The ultraviolet spectrum of the newantibiotic exhibits maxima at 218, 365 and 530 m in methanol, whereasmitomycin B exhibits maxima at 218, 320 and 537 m in methanol. The newantibiotic is active against bacteria such as Bacillus subtilis. The Rof the new antibiotic is 0.47 as against 0.64 for mitomycin B when runon paper strips as in Example 1.

EXAMPLE 3 The procedure of Example 1 is repeated using a series ofdifferent reagents. The results appear in the following table, the Rnumber being obtained by paper chromatography using the system describedin Example 1.

TABLE I Reagent Color U.V. AMeOlElm.X Rt

None 212 320 520 0. 74 Aniline 214 259 376 560 82 Isopropanolamine 247310 550 79 Cyclohexylamine. 365 9O 3 aminoquinoline 350 540 834-aminopyridine 350 525 84 Glycine 258 65 Piperazine 335 88 EXAMPLE 4The procedure of Example 2 is repeated with a number of other reagents.The results appear in Table II, the R numbers being taken with the samesystem as in Example 1.

One part of porfiromycin is dissolved in 1000 parts of water and a largeexcess of reagent in aqueous solution is added with stirring. Afterstanding protected from light for fifteen hours, the solution is driedfrom the frozen state to obtain a new colored crude antibiotic which ispurified by chromatography as in Example 1. The ultraviolet spectra ofthe new antibiotics exhibit characteristic maxima in methanol and havecharacteristic R values in the same system as in Example 1. In this samesystem porfiromycin has an R: of 0.68. The new antibiotics are activeagainst bacteria such as Bacillus subtilis.

This procedure is followed using different reagents. The results aretabulated in Table III, the R values being 6 EXAMPLE 10 A 2.0 mg. sampleof N-methyl mitomycin A is dissolved in 0.5 ml. of cold methanol andexcess sodium glycinate added. The reaction proceeds at 20 C. for 18measured in the same system as in Example 1. hours TABLE III 7 EXAMPLE11 Reagent Color U.v.).Me01I....x R: A 25 mg. sample of mitomycin A isdissolved in 1.5 ml. of methanol and cooled to 0 C. A ml. sample ofAniline Yellow 216 231 359 .54 N,N-dimethylethylenediamine is added andthe reaction gggg gg g g 212 gig; allowed to proceed at 5 C. for 18hours. The solvent is smino ri'dine'j: Yellow .70 removed. The productdissolves in benzene and re-pre- Plperazme 216 (240) 358 cipitates bythe addition of excess petroleum ether (30 60 C.). This crude sample ispurified chromatographical- EXAMPLE 6 1y to yield 22 mg. of pureproduct. One part of mitomycin C is dissolved in 1000 parts of water andan excess of reagent is added with stirring. The LE 12 reaction isallowed to stand for fifteen hours in the dark To a solution of 11 partsof 4 aminomethylpyridine and then the1Vent 1S drymg from frozen in 100parts of methanol is added a solution of 0.44 parts state to obtain thenew antibiotics as colored sollds. The of N benzyl mitomycin A in partsof methanoL The crude ant blotlcs are purlfied by chromat g P Y- h redsolution is allowed to stand for 20 hours at room temnew antlblotlcsshow characteristic ultraviolet spectra n peratura The Solvent isremoved by drying from the m6than01- M'ltomycm C an ultravlolet Spectrumm frozen state, and excess amine is removed by trituration methanol fSPOWS maxlma at 3 358 and 25 with petroleum ether (-60 C.) to give thecrude yel- The new antlblotlcs have characteinsuc values In the lowantibiotic which is then chromatographed as in Exsystempf Example the 9,mltomycn} C m l ample 1. The'ultraviolet spectrum of the new antibiotictern being 0.3 8. The new antibiotics are active against bacexhibitsmaxima at 363 and my in methanol Where terla as Bacillus as N-benzylmitomycin A exhibits maxima at 320 and ThlS procedure 15 iollowed uslngdlfierent reagents. The 30 525 In in methanol. The Ri of the newantibiotic is results are tabulated in Table IV, the R values beingmeas- 047 as compared with 027 for NJJBHZYI mitomycin A m the SameSystem asm Example When run as in Example 1. The new antibiotic isactive 'TABL IV against bacteria such as Bacillus subtilis.

Reagent Color U.V.)\Me0Hm1 R: EXAMPLE 13 Aniline Purple 215 232) 357 550.38 The Procedure of EXample 12 is repea ed using di-f- Morpholine Pink242 270 343 .33 ferent reagents with the N-benzyl mitomycin A. The dataobtained are shown in Table V below.

TABLE v Starting Material Reagent $3535 3g is? gr 15 N-B1e)nzy1mitomycinA N%11%Ib.6l.1.fii. g

r DO g-Anisidine. 3 4-ami11omethy1-pyridine...-- 47 N-methyl-benzylamine22 Ethyl p-amino-benzoate...-- 25 A LE 7 A number of tests were madewith some of the new A 5 mg. sample of N-methyl mitomycin A is dissolvedin 0.5 ml. of morpholine, and allowed to react at 20 C. for 18 hours. Asingle, 6.3 mg. peak is obtained when partition chromatographed 011 aCelite column using a heptane ethyl acetate methanol water systemEXAMPLE 8 EXAMPLE 9 A 27.1 mg. sample of N-methyl mitomycin A isdissolved in 5 ml. of methanol and to this is added 4.6 mg. ofethanolarnine in 0.5 ml. of methanol. The reaction is allowed to standfor 18 hours at 20 C. and then purified by chromatography. A 21.4 mg.homogeneous product is obtained.

antibiotics compared to one or more starting materials. Table VI showsinhibitory concentrations of the antibiotic porfiromycin and a newcompound prepared by reacting p'orfiromycin with p-toluidine.

TABLE VI Porfiromycin us p-toluidine Mycobaclerium smegmatis ATCC 607.0.31 0.31 Staphylococccus aureus ATCC 65381 0. 15 0. 15 Sarcirla lutcaATCC 9341 0. 62 0. 62 Bacillus subtills ATCC 6633 0.02 0. 02Streptococcus faecalis AIOO 8043. 1. 25 0. 62 Pseudomonas aeruginosaATOC 10145.. 5 10 Corynebacterium xerose NRRL B1397-. 0.15 0. 15Streptococcus pyogenes C203 0. 02 0.01 Streptococcus sp. 7 hem. No. 110. 15 8 Staphylococcus albus No. 69 0. 31 0131 Streptococcus sp., 3 hem.No. 2g 9g Staphylococcus aureus NY 104-.- 0: 31 0: 31 Bacillus cereusATCC 10702.. 0. O8 0. 08 Streptococcus pyogenes NY 5..-. 0.01 0.01Klebsiella pneumoniae media lab N 8. 0. 15 0. 15 Alcaliqenes faecalt'sA'ICO 10153 0. 62 1. 25 Escherichia coli No. 22 10 10 Klebsiellapneumoniae "A" Strain AD. 0. 31 3 The data of Table VI is summarized inrelative activity A vs the reaction product. The data appear in Tablesterms in Table VII. X and XI.

TABLE VII TABLE X Porfiromycln 5 Porfiromyein plus p-toluidine MitomyclnA M1tomycin A plus aniline llycohlmzclerium smegmaXsI lgICO 607..- 8 8 O62 l 25 slap 1/ ococcus aureus 5381 2 2 P D Sarcina lutea ATCC 9341 1 321 32 Miwbaclewm smegma ATCC 2. 5 5 Sacillus subtz'lis ATCC 6633 s s 10Staphylococcus aureus ATCC 6538P.-. 0. 31 0. s llreptococcus faecalisATCC 8043 1 2 Sarciha lulea ATCC 9341 0.04 0. 04 seudognonas aerugtnosaATRCLC 10145.. 1&2 l1 Bacillus subtilis ATCC 6633 0.31 (1)1 70mm;acterium xerosc NR B1397" 1 4 1 4 D firepmmus Mamas C203 1 2Streptococcusfaewlw, C 3 25 5 ltreptococcus sp., 7 hem. No. 11 8 8Pseudomonas aeruginosa ATCC 10145. 10 5 ltaphylococcus albus N o. 69 1/21/2 Proteus vulgaris ATCC 9484 1.25 itreptocoeeus sp., 5 hem. No. 80 4 4Escherichia coli ATCC 9637 10 ltaphylococcus aureus NY 104 1 1Salmonella gallinarum L-A-I- 604..---. 10 Zacillus cereus ATCC 10702 1616 Oory-nebacteriu-m xerose NRRL B1397 0.04 'ftgeglloggccus pyogenes N1;5.1 .-N 4 l1 Streptococcus pyogenes C203 0.15 0.01

e sze apnemnom'aeme ia ab 0. 8.- 4 1 llcaligmes faecalis ATCC10153.-..-. 1 1 Strepmcocws 25 l 0.08 Zschctt'chill coli No. 22. 4 2Staphylococcus albus No. 69 0.31 0.08 ilcbswlla pneumomac A Stram AD. 44 20 streptococcus Sp. 5 hem 80 1' 8gp Staphylococcus aureus NY 104..0.31 0.08 .gacillus cereus ATCC 11%(02- 0.01 t to u 10 n 5... 0.02Another test 2 macle 4 $0111? of the compolmfis Kz fl t lzlf wie hghzimedia lab. No 8. 1. 25 0. 31 iroduced by reacting with mltomycin B andhere, as m Alwlwems faemlis ATCC 10153 5 gg he case of Tables VI andVII, there is a corresponding 25 EscMTic/tia 22 able giving relativeactivity for two of the compounds. K760856110 lmwmoniawA" Strain 125 .hedata appear in Tables VIII and IX.

TABLE VIII Vlinimal Inhibitory Concentration of Mitomycln l3 Mitomycin BNew Antiobitics Prepared from Mito- Mitomycin B plus aniline plus ethyl-Mitomycin A myc'iu B and Standards eneimine 'ory'ltebaclerium zeroseNRRL B1397 0. 62 1. 25 0.08 0.04 taphylococcus aureus ATCC 65381. 5 1.25 1. 25 0.62 aroma lutea ATCC 9341 0. 62 0.31 0.15 0. 08 'acz'llussubtilisATCC 6633 1. 25 8 3% 0. 31 0.31 lreptococcus pyogenes C203 0. l50. 04 0.04 0.02 :reptococcus sp., 7 hem. No. 11 2. 5 0. 62 0.31 0. 62Eaphylococcus alhus N0. 69 5 1. 25 1. 25 0.15 .reptococcus sp., B hem.No. 80. 1. 25 0. 62 0.31 0.62 aphylococcus aureus NY 104--- 10 2. 5 2. 50. 31 acz'll'us ccreus ATCC 10702 5 0.62 1. 25 0. 62 seudomonasaeruglnosa ATCC 10145. 5 10 ireptococcus faecalis ATCC 8043 10 5p 1.25 1. 25 lreptococcus pyogenes NY 5 0.15 0.02 0. 04 0. 04 lebsiellapneumoniae media lab. N o. 8 2. 5 1. 25 0. 62 0.31 scherichia coll N o.22 10 1O lcaligenes faecalis ATCC 10153 1O 5 5 TABLE XI TABLE IXMitomycin A t it fN A tb' t' P d Mlt B M't B plus aniline 0 iv y o eW n1 1o res repare omyein 1 omycin from Mitomycin B Relative to plusAniline us Mycobactclmm Smegmatls ATCC 607 Mitomycin B ethylenelmmeStaphylococcus aureus ATCC 6538P 4 Sarcina lutea ATCC 9341 1ry'hebacterium zerose ljrRRL 121397.. 1/2 8 Bacillus subtilis ATCC 663316 iwjflif g ggfi tgggg fffii i Streptococcus faecalis ATCC 8043 2:cillus subtt'lis ATCC 6 63 3. i i Pseudomonas aeruginosa ATCC 10145 2gggi, ggggg ggfi ff i 4 8 Coryucbacterium xerose NRRL B1397 1/2zphtylococcus (IZUZShNO. 33. 3 Z 5 Streptococcus pyagenes C203 1/ 2 repococcus sp. em. 0. zphylgcaccus aural .3 NY 104 4 4 Streptococcus SP- 'YNO- cilus cereus ATCC 102%. 8 i Staphylococcus albus N0. 69 2 CU omonas(ZCTUQZI'LOSG 'eptococcusfaecalis ATCC 8043 8 streptococcus p- 80 16 5955 P11 9 0 5 5 5- 8 S 2 Staphylococcus aureus NY 104 4 71, mm 13.5.132522%. 2 70 Bacillus cereus ATCC 10702 64IIZZI'QWMSIMCIIZY'SATCG10153 1 2 Streptococcus pyogenes NY 5 2Klcbsiella pneumoniae medium lab. N0. 8 4 Tests were also made withmitomycin A and a reaction Alcaligencs faecalis ATCC 10153 2 oduct withaniline. As in Tables VI and VII, there is a Escherichia coli N0. 22 1/2rresponding table giving relative activity for mitocycin Klebsicllapneumoniae A Strain AD 8 In the foregoing tables wherever the inhibitionwas not 100% complete the notation p is used to denote that theinhibition was partial although nearly complete.

U. V. data, where given, occasionally show a wave length in parenthesis.This denotes a point of inflection and not a clear maximum.

EXAMPLE 14 In vivo activity of the novel antibiotics of this inventionwas demonstrated by experiments carried out as follows:

Unit test groups consist of or Carworth Farms CFl female mice averaging18 to 21 grams of body weight per mouse. Infections are produced byintraperitoneal injections of 0.5 milliliter volumes of a 10* trypticasesoy broth (TSP) dilution of a 5hour blood broth culture ofStaphylococcus aureus, strain Smith. The antibiotics in 0.2% aqueousagar are administered in single subcutaneous injection doses or singleoral tubing doses one-half hour after infection. Control mice receive0.2% aqueous agar without antibiotic.

The results obtained are shown in the table below.

TABLE XII Eflect on Mice oi the Product of N-Methyl Mitomycin A andMorpholine Administered in Slngle Subcutaneous Doses and Single OralTubing Doses One-half Hour after Standard Infection with Staphylococcusaureus, Strain Smith Survival after 6 Days Survivors/Total Dose, mgJkg.

Single Subcutaneous Single Oral Tubing Doses Doses Controls-435% (19/20)of infected nontreated control animals died in an average time of 1.0 to1.2 days.

TABLE XHI Effect on Mice of the New Antibiotics; The Product of N-MethylMitomycin A and fl-Dimethylaminoethylamine, and N-Methyl Mitomycin A andEthanolamine Administered in Single Subcutaneous Doses One Half Hourafter Standard Infection with Staphylococcus aureus, Strain SmithSurvival after 9 Days Survivors/Total Dose, rug/kg. N-Methyl MitomycinN-Methyl Mitomycin A plus fl-Dimethyl- A plus Ethanolamineaminoethylamine Controls.100% 20) of infected non-treated controlanimals died in an average time of 1.0 day.

TABLE XIV Efiect on Mice of the New Antibiotics Mitomycin A andfi-Dimethylaminoethylamine, and Mitomycin A and Aniline, Administered inSingle Subcutaneous Doses One-Half Hour after Standard Infection withStaphylococcus aureas, Strain Smith Survival after 14 DaysSurvivors/Total Dose, mg./kg.

A plus fi-Dimethyl- A plus Aniline aminoethylamine 1 0 What is claimedis: 1. A compound of the formula:

wherein R is selected from the. group consisting of phenyl loweralkylamino, N-lower alkyl phenyl lower alkylamino, lower dialkylaminolower alkylamino, carboxy lower alkylamino, lower alkyleneimino, cyclolower alkylamino, hydroxy lower alkylamino, pyridylamino, pyridyl loweralkylamino, quinolylamino, morpholino, piperazino and a moiety of theformula:

@NEL

wherein R is selected from the group consisting of hydrogen, halogen,lower alkyl, lower alk-oxy and lower carboalkoxy; R is selected from thegroup consisting of hydrogen and methyl; and R is selected from thegroup consisting of hydrogen, lower alkyl, and phenyl lower alkyl.

2. A compound of the formula:

4. A compound of the formula:

1 1 1 2 6. A compound of the formula: 9. A compound of the formula:

ll 0 Q H N CHZOCNHZ 0 CH3 5 aC H N 0 NH 7 A com ound of the formula 10 p10. A compound of the formula:

0 CHzCEz [I El H H O HN N CHZOCNH: 20 N H OH 0% OH OH 00113 z z NH2 2 2CHzCz OH H30 30 N 0 NH ll 0 NCH3 8. A compound of the formula:

0 References Cited 2 H 0 1] UNITED STATES PATENTS N CH2OCNH2 OH3,306,821 2/1967 Schoeder 167-65 2 H N ALEX MAZEL, Primary Examiner.

0 NCHa H. R. JILES, Examiner.

M. U. OBRIEN, J. TOVAR, Assistant Examiners.

1. A COMPOUND OF THE FORMULA:
 2. A COMPOUND OF THE FORMULA: